CN112980873A - Protein related to plant type and coding gene and application thereof - Google Patents

Abstract

The invention discloses a protein related to plant types and a coding gene and application thereof. The invention provides application of DHT1 protein or related biological materials thereof in regulation and control of plant grain development. The related biological material is a nucleic acid molecule capable of expressing DHT1 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule; the DHT1 protein is a protein shown in SEQ ID No.1 or a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues, has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology in sequence and has the same function, or is a fusion protein obtained by connecting a label at the N end and/or the C end of the protein. Experiments prove that the DHT1 and the encoding gene thereof can regulate the plant type of the plant, and have important application value for effectively regulating the plant type of the plant by utilizing the gene resource through genetic breeding and genetic engineering methods.

Description

Protein related to plant type and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a protein related to plant types of plants, and a coding gene and application thereof.

Background

Rice, as an important food crop, lives over one third of the world's population. With the growing population, the reduction of the cultivated land area and the deterioration of the environment, rice production is under greater and greater pressure in terms of ensuring yield. The plant type of rice is an important agronomic trait related to yield, and mainly depends on several aspects of plant height, leaf type, tillering number, tillering angle, spike morphology and the like. Therefore, the discovery and the utilization of the related gene for controlling the rice plant type have important significance for rice breeding and production.

Until now, most of cloned plant height genes of rice are related to the metabolism or signal transduction of hormones such as gibberellin, brassinolide and strigolactone, for example, a half-dwarf gene sd1 widely applied in the current production encodes gibberellin synthesis oxidase. The research also finds that other genetic network genes such as hormone balance and interaction, RNA editing, cell wall development and the like are all involved in the formation of the plant height, and the formation of the plant height of the rice can be regulated and controlled by a complex genetic network.

Post-transcriptional regulation is one of the important regulatory mechanisms in the process of gene expression. In eukaryotes, the post-transcriptional RNA processing modification process mainly includes: splicing of mRNA precursor (pre-mRNA splicing), capping of the 5 'end of mRNA (capping), polyadenylation of the 3' end of mRNA to a Poly A tail (polyadenylation), transport of mRNA, stabilization and translation of mRNA, etc. RNA-binding proteins (RBPs) participate in various RNA processing and modifying processes to regulate the expression of specific transcripts, so that the normal growth and development, stress resistance and other life processes of plants are influenced. At present, the protein in the plant is involved in regulating and controlling a plurality of physiological and biochemical processes such as seed germination, photosynthesis, various adversity stress responses, flowering, hormone signal conduction and the like, but the regulation and control of the plant type of the plant are unknown.

Disclosure of Invention

The invention aims to provide a protein related to plant types and a coding gene and application thereof.

In a first aspect, the present invention claims any one of the following uses of the DHT1 protein or related biological material thereof;

p1, regulating and controlling plant types;

p2, regulating and controlling the plant height;

p3, regulating and controlling the plant ear length;

p4, regulating and controlling the grain width of the plant;

p5, regulating and controlling the tillering number of the plant.

Wherein, the related biological material can be a nucleic acid molecule capable of expressing the DHT1 protein, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the nucleic acid molecule.

The expression cassette refers to a DNA capable of expressing DHT1 in a host cell, and the DNA may include not only a promoter for initiating transcription of DHT1 gene, but also a terminator for terminating transcription of DHT 1. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: ubiquitin gene ubiqiutin promoter (pUbi); the constitutive promoter of cauliflower mosaic virus 35S; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No.5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I)⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al Genes Dev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

Constructing a recombinant expression vector containing the ZmGW3 gene expression cassette. The plant expression vector can be binary Agrobacterium vector or Gateway system vector, such as pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pGWB411, pGWB412, pGWB405, pCAMBIA1391-Xa or pCAMBIA 1391-Xb. When ZmGW3 is used to construct recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV)35S promoter, ubiquitin gene ubiqiutin promoter (pUbi) and the like can be added before the transcription initiation nucleotide, and can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. The bacteria can be derived from Escherichia (Escherichia), Erwinia (Erwinia), Agrobacterium (Agrobacterium) such as Agrobacterium tumefaciens EHA105, Flavobacterium (Flavobacterium), Alcaligenes (Alcaligenes), Pseudomonas (Pseudomonas), Bacillus (Bacillus), etc.

The DHT1 protein can be any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) the amino acid sequence shown in SEQ ID No.1 is substituted and/or deleted and/or added by one or more amino acid residues and is derived from the protein with the same function of rice;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and derived from rice having the same function;

(A4) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above protein, the homology of 95% or more may be at least 96%, 97%, 98% identity. The homology of 90% or more may be at least 91%, 92%, 93%, 94% identity. The homology of 85% or more may be at least 86%, 87%, 88%, 89% identity. The homology of 80% or more may be at least 81%, 82%, 83%, 84% identity.

The expression level and/or activity of the DHT1 protein in the plant is reduced, and the plant height of the plant is reduced, the ear length of the plant is shortened, the grain width of the plant is shortened, and/or the tillering number of the plant is increased.

In a second aspect, the present invention claims the use of a substance capable of reducing the expression level and/or activity of DHT1 protein in a plant, in any one of (a1) - (a 4):

(a1) reducing the plant height of the plant;

(a2) reducing the ear length of the plant;

(a3) reducing the grain width of the plant;

(a4) increasing the number of tillers of the plant.

The DHT1 protein can be any one of the proteins shown in the above (A1) - (A4).

In a specific embodiment of the present invention, the "substance capable of reducing the expression level and/or activity of DHT1 protein in a plant" is specifically a DNA fragment represented by formula I below or an interference vector containing a DNA fragment represented by formula I below;

SEQ_{forward direction}-X-SEQ_{Reverse direction}(I)；

Said SEQ_{Forward direction}Is a partial fragment of SEQ ID No.2 or the full length thereof;

said SEQ_{Reverse direction}And the sequence of SEQ_{Forward direction}Is complementary in reverse direction;

said X is said SEQ_{Forward direction}And said SEQ_{Reverse direction}In the sequence, the X and the SEQ_{Forward direction}And said SEQ_{Reverse direction}Are not complementary.

Further, said SEQ_{Forward direction}Is the nucleotide sequence of 650-890 position of SEQ ID No.2, which is SEQ ID No. 4.

The nucleotide sequence of the DNA fragment shown in the formula I is SEQ ID No.5, the nucleotide sequence from the 1 st to the 241 th positions in the SEQ ID No.5 is the nucleotide sequence from the 650 th-through 890 th positions in the SEQ ID No.2, the nucleotide sequence from the 269 th-through 1264 th positions is the nucleotide sequence of Arabidopsis FAD2 intron, and the nucleotide sequence from the 1438 th-through 1678 th positions is the nucleotide sequence reverse complementary to the nucleotide sequence from the 650 th-through 890 th positions in the SEQ ID No. 2.

In a third aspect, the invention claims a method for cultivating plants with reduced plant height, shortened ear length, reduced kernel width and/or increased tiller number.

The method for cultivating the plant with reduced plant height, shortened ear length, reduced kernel width and/or increased tiller number, which is claimed by the invention, can comprise the step of reducing the expression amount and/or activity of DHT1 protein in a receptor plant.

The DHT1 protein can be any one of the proteins shown in the above (A1) - (A4).

The method can be realized by means of hybridization or by means of transgenosis.

In a fourth aspect, the invention claims a method for cultivating transgenic plants with reduced plant height and/or shortened ear length and/or reduced kernel width and/or increased tiller number.

The method for cultivating the transgenic plant with reduced plant height, shortened ear length, reduced kernel width and/or increased tiller number, which is claimed by the invention, can comprise the following steps: inhibiting and expressing a nucleic acid molecule capable of expressing DHT1 protein in a receptor plant to obtain a transgenic plant; compared with the acceptor plant, the transgenic plant has the advantages of reduced plant height, shortened spike length, reduced kernel width and/or increased tiller number.

The DHT1 protein can be any one of the proteins shown in the above (A1) - (A4).

In the method, the inhibition of the expression of the nucleic acid molecule capable of expressing the DHT1 protein in the recipient plant may be achieved by any means capable of achieving this.

In a specific embodiment of the present invention, the suppression of expression of the nucleic acid molecule capable of expressing the DHT1 protein in the recipient plant is achieved by introducing into the recipient plant an interference vector comprising a DNA fragment represented by formula I below;

SEQ_{forward direction}-X-SEQ_{Reverse direction}(I)；

Said SEQ_{Forward direction}Is a partial fragment of SEQ ID No.2 or the full length thereof;

said SEQ_{Reverse direction}And the sequence of SEQ_{Forward direction}Is complementary in reverse direction;

said X is said SEQ_{Forward direction}And said SEQ_{Reverse direction}In the sequence, the X and the SEQ_{Forward direction}And said SEQ_{Reverse direction}Are not complementary.

Further, said SEQ_{Forward direction}Is the nucleotide sequence of 650-890 position of SEQ ID No.2, which is SEQ ID No. 4.

The nucleotide sequence of the DNA fragment shown in the formula I is SEQ ID No.5, the nucleotide sequence from the 1 st to the 241 th positions in the SEQ ID No.5 is the nucleotide sequence from the 650 th-through 890 th positions in the SEQ ID No.2, the nucleotide sequence from the 269 th-through 1264 th positions is the nucleotide sequence of Arabidopsis FAD2 intron, and the nucleotide sequence from the 1438 th-through 1678 th positions is the nucleotide sequence reverse complementary to the nucleotide sequence from the 650 th-through 890 th positions in the SEQ ID No. 2.

In a specific embodiment of the present invention, the interference vector containing the DNA fragment represented by the following formula I is specifically pLHRNAi-DHT 1. The pLHRNAi-DHT11 is a recombinant vector obtained by replacing a DNA sequence between SacI and SnaBI recognition sites (recognition sequences) of pLHRNAi with a DNA fragment (SEQ ID No.5) shown in a formula I.

In the above method, the interference vector containing a DNA fragment represented by the following formula I is introduced into the recipient plant, and specifically may be: plant cells or tissues are transformed by conventional biological methods using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and the transformed plant tissues are grown into plants.

In the above methods, the transgenic plant is understood to include not only the first to second generation transgenic plants but also the progeny thereof. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

In each of the above aspects, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA, and the like.

In the present invention, the nucleic acid molecule capable of expressing the DHT1 protein may specifically be any of the following:

(B1) a DNA molecule shown as SEQ ID No.2 or SEQ ID No. 3;

(B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes said DHT1 protein;

(B3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence defined in any one of (B1) - (B2) and encodes the DHT1 protein.

In the above nucleic acid molecule, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In the above nucleic acid molecules, homology means the identity of nucleotide sequences. The identity of the nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of nucleotide sequences, a value (%) of identity can be obtained.

In the above nucleic acid molecule, the homology of 95% or more may be at least 96%, 97%, 98% identity. The homology of 90% or more may be at least 91%, 92%, 93%, 94% identity. The homology of 85% or more may be at least 86%, 87%, 88%, 89% identity. The homology of 80% or more may be at least 81%, 82%, 83%, 84% identity.

In a fifth aspect, the invention claims the use of the method described in the third or fourth aspect hereinbefore in plant breeding.

In the above aspects, the plant is a monocot or a dicot.

Further, the plant is a gramineous plant.

Further, the plant is a plant of the genus oryza, such as rice.

In a particular embodiment of the invention, the plant is in particular the rice variety Kitaake.

Experiments prove that the plant type related protein DHT1 and the encoding gene thereof can regulate and control the plant type of plants, particularly the plant height, the spike length, the grain width and the tillering number. Inhibiting the expression of the plant type related protein DHT1 encoding gene can obviously reduce the plant height, the ear length, the grain width and the tillering number of the plant, and proves that the plant type related protein DHT1 and the gene thereof play an important role in regulating and controlling the plant height, the ear length, the grain width and the tillering number of the plant. The invention not only provides a basis for further clarifying the molecular mechanism of plant types of plants, but also provides new gene resources and breeding resources for plant breeding. The plant type related protein DHT1, the coding gene thereof and the nucleic acid molecule for inhibiting the gene expression can be used for cultivating plants with reduced plant height; the transgenic plant with reduced DHT1 gene expression, which is obtained by the invention, can be used as a new plant seed material for researching the molecular mechanism of plant dwarfing and finding more genes for regulating and controlling the plant height development of plants. The invention has important application value for effectively regulating and controlling the plant type of the plant by utilizing the gene resource through genetic breeding and genetic engineering methods.

Drawings

FIG. 1 shows the transcript level detection of DHT1 gene in wild type rice and transgenic interfering lines. Indicates that the difference was very significant at the P <0.01 level.

FIG. 2 is a phenotypic observation of RNA interference transgenic rice with reduced expression level of DHT1 gene. A is the phenotype of a wild rice plant and a positive interference plant; b is the spikelet phenotype of the rice wild type plant and the positive interference plant; c is the phenotype of the rice wild type plant and the positive interference plant seed; d is the comparison of the height of the wild type and the positive interference positive plant; e is the comparison of the wild type and positive interference positive plant tillering; f is the comparison of the wild type and the ear length of the positive interference positive plant; g is the comparison of the grain width of the wild type and positive interference positive plants. In the figure, Ri1 represents an RNAi-1 plant, and Ri2 represents an RNAi-2 plant. Indicates that the difference was very significant at the P <0.01 level.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

Rice Kitaake (also known as wild type rice, abbreviated WT) in the following examples: as described in GaoH, ZhengXM, WanJM, et al, Ehd4 encodes aNovel and Oryza-gene-specific regulator of photo-electronic flow in rice PLOS GENET.2013,9(2) e 1003281. In this document "Kita-ake". The biological material is available to the public from the institute of crop science, academy of agricultural sciences, and is used only for repeating the relevant experiments of the present invention, and is not used for other purposes.

The expression vector pLHRNAi used in the following examples is pLHRNAi in Chinese patent 201110055864.X (the specific construction method steps of the vector are explicitly described in the patent), the public can obtain the biological material from the research institute of crop science of Chinese academy of agricultural sciences, and the biological material is only used for repeating relevant experiments of the invention and cannot be used for other purposes.

The Agrobacterium used in the examples described below is Agrobacterium tumefaciens EHA105(Agrobacterium tumefaciens EHA105), described in the following literature, New Agrobacterium heler plasmids for gene transfer to plants, hood, ElizabethE; gelvin, Stanton B; melchers, LeoS; hoekema, andre. transgenic research,2(4): p.208-218(1993), which is publicly available from the institute of crop science, academy of agricultural sciences, and is used only for repeating the experiments related to the present invention, and is not used for other purposes.

Example 1 plant type-related protein DHT1 can regulate and control plant height, panicle length and grain width of rice

This example provides a plant-type related protein derived from rice Kitaake (Oryza sativa var. Kitaake), named DHT1, in which the amino acid sequence of DHT1 is SEQ ID No.1, the coding sequence (i.e., CDS sequence) of DHT1 gene is SEQ ID No.2, and the genomic sequence of DHT1 gene is SEQ ID No. 3.

Construction of RNA interference vector for reducing DHT1 gene expression level

1. Obtaining of interference fragment for DHT1 gene expression

(1) Total RNA of 14-day seedlings of rice Kitaake (Oryzasativa) was extracted using an RNAprep plant total RNA extraction kit (Tiangen Biochemical technology, Beijing) Co., Ltd.) and reverse-transcribed to obtain cDNA.

(2) Performing PCR amplification by using the cDNA obtained in the step (1) as a template and a primer pair consisting of DHT1-sense-F and DHT1-sense-R to obtain a fragment 1(SEQ ID NO: 1)_{Forward direction}As shown in SEQ ID No. 4).

DHT1-sense-F：5'-TTCTGCACTAGGTACCAGGCCTGCTGGTTCTGCGTCGTCTTCAC-3'；

DHT1-sense-R：5'-CTGACGTAGGGGCGATAGAGCTCCCATCAAAGTTCCTTGTTGGCTC-3'。

(3) Using the cDNA obtained in the step (1) as a template, and carrying out PCR amplification by using a primer consisting of DHT1-antisense-F and DHT1-antisense-R to obtain a fragment 2(SEQ ID NO: 2)_{Reverse direction}As shown in the reverse complement of SEQ ID No. 4).

DHT1-antisense-F：5'-CGGGGATCCGTCGACTACCTGGTTCTGCGTCGTCTTCAC-3'；

DHT1-antisense-R：5'-AGGTGGAAGACGCGTTACCCATCAAAGTTCCTTGTTGGCTC-3'。

2. Construction of DHT1 Gene RNA interference vector (recombinant expression vector pLHRNAi-DHT1)

(1) The expression vector pLHRNAi is digested by restriction endonuclease SacI to obtain a linear expression vector pLHRNAi, and the linear fragment is recovered. Integrating the fragment 1 obtained in the step 1 (2) to a linear expression vector pLHRNAi (the concrete method refers to the clone infusion kit instruction) by adopting a homologous recombination directional cloning method to obtain a homologous recombination product 1, then transferring the homologous recombination product 1 to escherichia coli DH5 alpha competent cells, culturing overnight at 37 ℃, and marking the obtained recombination vector with a correct sequence as pLHRNAi-sense-DHT 1.

(2) The recombinant vector pLHRNAi-sense-DHT1 was digested with restriction enzyme SnaBI to obtain the linear vector pLHRNAi-sense-DHT1, which was recovered. Integrating the fragment 2 obtained in the step 1 (3) to a linear vector pLHRNAi-sense-DHT1 by adopting a homologous recombination directional cloning method (the concrete method refers to the clone infusion kit instruction), obtaining a homologous recombination product 2, transferring the homologous recombination product 2 to an escherichia coli DH5 alpha competent cell, culturing overnight at 37 ℃, and marking the obtained recombination vector with a correct sequence as pLHRNAi-DHT 1.

(3) Sequencing is carried out on the recombinant vector pLHRNAi-DHT1, and the result shows that the recombinant vector pLHRNAi-DHT1 is formed by inserting a double-stranded DNA fragment shown by a nucleotide sequence 650 to 890 from the 5 'end of SEQ ID No.2 into a SacI enzyme cutting site of an expression vector pLHRNAi in the forward direction, and inserting a double-stranded DNA fragment which is reversely complementary to the double-stranded DNA fragment shown by a nucleotide sequence 650 to 890 from the 5' end of SEQ ID No.2 into a SnaBI enzyme cutting site, so that a DNA sequence between the SacI and the SnaBI recognition sites (recognition sequences) of the pLHRNAi is successfully replaced by the DNA fragment shown by SEQ ID No. 5.

The nucleotide sequence at the 1-241 th site in the SEQ ID No.5 is the nucleotide sequence at the 650-890 th site in the SEQ ID No.2, the nucleotide sequence at the 269-1264 th site is the nucleotide sequence of Arabidopsis FAD2 intron, and the nucleotide sequence at the 1438-1678 th site is the nucleotide sequence of the 650-890 th site in the SEQ ID No.2 which is reversely complementary.

Constructing RNAi interference transgenic plant with reduced DHT1 gene expression level and identifying transgenic plant

1. Construction of transgenic plants

The rice Kitaake is mediated and transformed by a recombinant vector pLHRNAi-DHT1 through Agrobacterium tumefaciens EHA105, and an empty vector control plant is constructed by utilizing the expression vector pLHRNAi according to the same method, and the specific method is as follows:

(1) and (3) introducing the recombinant vector pLHRNAi-DHT1 obtained in the step one into the Agrobacterium tumefaciens EHA105 by a heat shock method to obtain the recombinant Agrobacterium tumefaciens EHA105 containing the recombinant vector pLHRNAi-DHT 1. The recombinant Agrobacterium tumefaciens EHA105 was cultured at 28 ℃ for 16 hours, and the cells were collected. The bacterial cells were diluted with N6 liquid medium (Sigma, catalog No. C1416) containing 100. mu.M acetosyringone to obtain diluted bacterial solution, OD600 of which was about 0.5.

(2) Mixing mature embryogenic callus of rice Kitaake cultured for one month with the diluted bacterial liquid obtained in step (1), infecting for 30min, drying the surface bacterial liquid of callus with filter paper, transferring into N6 solid co-culture medium (N6 mixed medium formula is potassium nitrate (2800mg/L), ammonium sulfate (463mg/L), potassium dihydrogen phosphate (400mg/L), magnesium sulfate (MgSO 2)₄·7H₂O) (185mg/L), calcium chloride (CaCl)₂·2H₂O) (165mg/L), disodium ethylene diamine tetraacetate (37.3mg/L), ferrous sulfate (FeSO)₄·7H₂O) (27.8mg/L), manganese sulfate (MnSO)₄·H₂O) (4.4mg/L), zinc sulfate (ZnSO)₄·7H₂O) (1.5mg/L), boric acid (1.6mg/L), potassium iodide (0.8mg/L), vitamin B1 (thiamine hydrochloride) (1.0mg/L), vitamin B6 (pyridoxine hydrochloride) (0.5mg/L), nicotinic acid (0.5mg/L), glycine (2.0mg/L), sucrose (20000 mg/L). Weighing 24.1g of N6 mixed culture medium, heating and stirring to dissolve in 1000ml of distilled water, adjusting pH to 5.8 with sodium hydroxide, autoclaving at 115 deg.C for 20 min to obtain N6 solid co-culture medium), co-culturing at 24 deg.C for 3d to obtain co-culture mediumAnd (5) culturing the treated callus.

(3) The callus after the co-culture treatment of step (2) was inoculated on N6 solid selection medium (a medium obtained by adding hygromycin to N6 solid medium, the mass concentration of hygromycin in the N6 solid selection medium being 150mg/L) containing hygromycin at a mass concentration of 150mg/L for the first selection.

(4) And (3) picking the healthy callus on the 16 th day from the first screening, transferring the healthy callus to an N6 solid screening culture medium (a culture medium obtained by adding hygromycin to an N6 solid culture medium, wherein the mass concentration of the hygromycin in the N6 solid screening culture medium is 200mg/L) containing the hygromycin, culturing, and carrying out secondary screening, wherein the secondary screening is carried out once every 15 days and is carried out for 1 time in total, and the obtained healthy callus is the resistant callus.

(5) And (3) transferring the resistant callus obtained in the step (4) to a differentiation medium (the differentiation medium: 6-BA2mg, NAA0.2mg, N6 mixed medium 4g, hydrolyzed casein 1g, inositol 0.1g, sucrose 25g, sorbitol 2.4g, agar powder 7g and deionized water to supplement 1L) containing hygromycin with the mass concentration of 150mg/L for differentiation culture, culturing at 24 ℃ for 45d (the height of the overground part of the plant is about 15cm), opening the bottle mouth, hardening seedlings for 3 days, and then transplanting to a greenhouse for culture, namely a transferred pLHRNAi-DHT1 plant (T0 generation).

2. RNAi interference transgenic plant PCR identification with reduced DHT1 gene expression level

Extracting genome DNA of T0 generation seedlings of the transgenic pLHRNAi-DHT1 plant and seedlings of rice Kitaake plant (abbreviated as WT) obtained in the step 1, and performing PCR molecular detection by using primers 1390-F (5'-TGCCTTCATACGCTATTTATTTGC-3') and FAD2-R (5'-GAAGCGACGGACCTGGAGAT-3') to identify positive seedlings to obtain plants of 382bp PCR products, namely positive transgenic plants (the two primers are designed according to the sequence of the pLHRNAi interference vector)_{Is just}). Therefore, the pair of primers is used for amplifying the sequence of the interference vector so as to verify whether the vector is transferred into a plant body, and the rice Kitaake plant (wild type) cannot obtain a 382bp PCR product. Randomly taking two positive transgenesThe plants are named as a pLHRNAi-DHT1 transferred plant RNAi-1 (RNAi-1 plant for short) and a pLHRNAi-DHT1 transferred plant RNAi-2 (RNAi-2 plant for short), respectively.

Third, RNAi interference of transgenic plant with reduced DHT1 gene expression level interferes with identification of DHT1 gene expression level

Respectively extracting RNA of the RNAi-1 plant, the RNAi-2 plant and the rice Kitaake plant (wild type) leaf obtained in the second step, setting an internal reference as Ubiquitin, and carrying out fluorescent quantitative PCR by using internal reference primers UBI-F and UBI-R and DHT1 gene specific quantitative primers DHT1-qRT-F and DHT1-qRT-R to detect the change of the expression level of the DHT1 gene of each plant. The results show (fig. 1) that compared with the expression level of DHT1 gene in rice Kitaake (wild type), the expression levels of DHT1 gene in RNAi-1 plant and RNAi-2 plant were significantly reduced, and there was no significant difference in the expression levels of DHT1 gene in empty vector control plant and rice Kitaake. The primers are as follows:

UBI-F：5’-GCTCCGTGGCGGTATCAT-3’；

UBI-R：5’-CGGCAGTTGACAGCCCTAG-3’；

DHT1-qRT-F：5’-TTTGGGACACAGGGCTTTGCAG-3’；

DHT1-qRT-R：5’-GCTTGAACACCCGCTGCATTTG-3’。

RNAi interference of transgenic plants with reduced DHT1 gene expression level

And (3) respectively planting the RNAi-1 plant, the RNAi-2 plant, the empty carrier control plant and the rice Kitaake plant (wild type) obtained in the step two in a consequent experimental base of the institute of crop science of the Chinese academy of agricultural sciences, and observing the phenotypic difference of each plant in the whole growth period. The observation results are shown in FIG. 2, compared with the rice Kitaake (wild type) plants, the RNAi-1 plants and the RNAi-2 plants have the phenotypes of short plant height, short panicle length, small grain width and increased tillering, and the average plant heights of the rice Kitaake (wild type), the RNAi-1 plants and the RNAi-2 plants are respectively 70 +/-0.95 cm, 46.40 +/-0.92 cm and 42.00 +/-0.47 cm in the mature period of rice; the average tillering of the rice Kitaake (wild type), the RNAi-1 plant and the RNAi-2 plant is 22.90 +/-1.95, 49.10 +/-3.52 and 57.20 +/-3.07 respectively; the average ear lengths of the rice Kitaake (wild type), the RNAi-1 plant and the RNAi-2 plant are respectively 13.31 +/-0.11, 10.47 +/-0.17 and 9.23 +/-0.13; the average grain widths of the rice Kitaake (wild type), RNAi-1 plant and RNAi-2 plant are respectively 3.68 +/-0.02, 3.13 +/-0.02 and 2.98 +/-0.03. The height of the empty vector control plant and the rice Kitaake plant has no obvious difference. Thus proving that the DHT1 gene participates in controlling the growth and development of rice plant types.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> institute of crop science of Chinese academy of agricultural sciences

<120> plant type-related protein, and coding gene and application thereof

<130> GNCLN210987

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 515

<212> PRT

<213> Oryza sativa L.

<400> 1

Met Ala Pro Lys Lys Arg Lys Ala Asp Pro Ala Glu Ser Pro Val Ala

1 5 10 15

Ser Ser Glu Ala Gly Ala Gly Thr Asn His His Gln Glu Thr Pro Ser

20 25 30

Ser Glu Leu Lys Pro Arg Gly Thr Ile Tyr Phe Pro Ile Thr Asp Asp

35 40 45

Pro Pro Glu Pro Ser Ala Glu Gly Gly Ala Glu Gly Glu Asp Gly Ala

50 55 60

Gly Gly Asp Asp Asp Glu Glu Asp Ile Ala Lys Leu Leu Glu Pro Leu

65 70 75 80

Ser Arg Glu Gln Leu Val Ala Leu Leu Arg Thr Ala Ala Glu Thr Thr

85 90 95

Pro Ala Thr Met Ala Ala Val Arg Arg Ala Ala Glu Ala Asp Pro Ala

100 105 110

Ser Arg Lys Leu Phe Val His Gly Leu Gly Trp Gly Ala Gly Ala Asp

115 120 125

Asp Leu Arg Ser Ala Phe Ser Arg Phe Gly Glu Leu Glu Asp Cys Arg

130 135 140

Val Ile Ser Asp Lys Gln Ser Gly Arg Ser Lys Gly Tyr Gly Phe Val

145 150 155 160

Leu Phe Arg Ser Arg Arg Ser Ala Leu Arg Ala Leu Arg Arg Pro Gln

165 170 175

Leu Gln Ile Gly Gly Arg Leu Ala Phe Cys His Leu Ala Ala Ser Gly

180 185 190

Pro Ala Pro Pro Thr Ser Gln Ser Gln Asn Pro Ser Ser Asn Thr Asn

195 200 205

Ala Asn Ser Gly Ala Ala Asn Asn Ala Gly Ser Ala Ser Ser Ser Gln

210 215 220

Pro Asp Asn Met Gln Arg Lys Ile Phe Val Gly Asn Val His Ala Asp

225 230 235 240

Val Asp Val Asp Arg Leu Tyr Glu Tyr Phe Ser Gln Phe Gly Glu Ile

245 250 255

Glu Glu Gly Pro Leu Gly Phe Asp Lys Thr Thr Gly Lys Pro Lys Gly

260 265 270

Phe Ala Leu Phe Val Tyr Lys Ser Val Glu Ser Ala Arg Arg Ala Leu

275 280 285

Glu Glu Pro Thr Arg Asn Phe Asp Gly Lys Met Leu Asn Val Gln Lys

290 295 300

Ala Ile Asp Gly Arg Thr Lys Asn Thr Pro Gly Met Asn Ala Asn Ser

305 310 315 320

Asn Pro Ser Gly Thr Ala Ala Ser Ala Ala Ala Ala Ala Ala Ala Ala

325 330 335

Gln Met Thr Ala Pro Ala Thr Ala Ala Ile Thr Pro Tyr Asp Ala Ser

340 345 350

Ala Tyr Gly Ala Thr Ala Val Pro Asp Leu Gly Tyr Ala Gln Gln Ala

355 360 365

Ala Met Leu Gly Leu Gly Ala Gln Gln Gln Ala Phe Ala Gln Pro Asn

370 375 380

Ala Ala Met Leu Ala Met Met Ala Ala Ala Met Gln Asn Pro Ala Met

385 390 395 400

Leu Ala Thr Leu Asn Pro Ala Phe Ala Ala Ala Ala Leu Gly Ala Gly

405 410 415

Gly Gln Gln Val His Ala Ala Gly Ile Pro Gly Phe Gly Ala Gln Gly

420 425 430

Phe Gly Thr Gln Gly Phe Ala Ala Gly Ala Ala Ala Phe Pro Asn Ala

435 440 445

Ala Gly Val Gln Ala Pro Pro Gly Phe Gln Gly Pro Pro Gly Phe Gln

450 455 460

Thr Ser Ala Gly Phe Gln Val Gly Gln Ala Ala Ser Gln Ala Gly Thr

465 470 475 480

Ala Ala Ala Ala Ala Ala Gly Ala Ala Gly Tyr Gln Ala Ala Ala Ala

485 490 495

Gly Gln Gly Gln Val Pro Gly Thr Gln Ile Gly Gly Ala Gly Phe Gln

500 505 510

Gly Gly Phe

515

<210> 2

<211> 1548

<212> DNA

<213> Oryza sativa L.

<400> 2

atggctccga agaagcgaaa ggcggatccg gcggagtccc cggtagcctc gtcggaggcc 60

ggcgccggca ccaaccacca ccaggagacg ccctcctcgg agctcaagcc ccgggggacc 120

atctacttcc ccatcaccga cgaccccccg gagccgagcg cagagggcgg cgccgagggg 180

gaggacggcg ccggagggga cgacgatgag gaggacatcg cgaagctgct cgagcccctg 240

tcgcgggagc agctggtggc gctgctccgc acggccgcgg agactacccc cgcgacgatg 300

gcggccgtgc ggcgcgcggc ggaggccgac cccgcgagca ggaagctctt cgtccacggc 360

ctcggctggg gcgccggagc cgacgacctc cgctccgcgt tctcccgctt cggcgagctc 420

gaggactgcc gtgtcatctc cgacaagcaa tctggcagat ctaagggcta cggcttcgtc 480

ctcttccgct cccgccgctc cgcgctccgc gccctccgcc gcccccagct ccaaatcggc 540

ggccgccttg ctttctgcca tctcgctgcc tcgggtcctg cccctccgac ctcccagtcc 600

cagaacccta gctccaacac caacgccaac tccggtgcgg ccaataacgc tggttctgcg 660

tcgtcttcac aacctgacaa catgcaacgc aaaatctttg ttggtaacgt gcatgctgat 720

gtcgatgttg accgcctgta tgagtacttc tcgcaatttg gtgagattga ggaggggcca 780

ttgggatttg acaagaccac tggcaagcct aaggggtttg cgctgttcgt ctacaagtca 840

gtggagagcg cccgccgtgc gctggaggag ccaacaagga actttgatgg caaaatgctc 900

aatgtgcaga aggctataga tggcaggacc aagaacacac ctgggatgaa tgcaaattct 960

aaccctagtg gtaccgctgc ctcggcagca gctgctgctg cagctgcaca gatgactgct 1020

cctgctactg ctgcaattac accgtatgat gcatcagctt atggtgctac tgccgttcct 1080

gacttgggtt atgcgcagca agcggctatg ttgggattgg gtgcacagca gcaggcgttt 1140

gcacaaccca atgctgcaat gcttgccatg atggcagcag ctatgcaaaa cccagctatg 1200

ctcgcaacgc tgaaccctgc ttttgctgct gctgcattgg gtgctggggg ccagcaggtg 1260

cacgcggctg gtattccagg ttttggagct cagggttttg ggacacaggg ctttgcagca 1320

ggtgccgccg cttttccaaa tgcagcgggt gttcaagctc ctcctggttt tcagggtccg 1380

cctgggttcc agacttctgc tgggtttcag gttggccaag cagcttcaca agcgggtact 1440

gctgcggctg ctgccgccgg tgctgctggt tatcaggctg ctgctgctgg gcagggccaa 1500

gtgcccggaa cgcaaattgg gggtgctggt tttcagggtg gattttga 1548

<210> 3

<211> 4048

<212> DNA

<213> Oryza sativa L.

<400> 3

tctctctctc tctctctctc tctctcccgt gtccgcaacc gcaaatcccc atttggtctc 60

acctaaaacc ctagctcgct cacccatggc tccgaagaag cgaaaggcgg atccggcgga 120

gtccccggta gcctcgtcgg aggccggcgc cggcaccaac caccaccagg agacgccctc 180

ctcggagctc aagccccggg ggaccatcta cttccccatc accgacgacc ccccggagcc 240

gagcgcagag ggcggcgccg agggggagga cggcgccgga ggggacgacg atgaggagga 300

catcgcgaag ctgctcgagc ccctgtcgcg ggagcagctg gtggcgctgc tccgcacggc 360

cgcggagact acccccgcga cgatggcggc cgtgcggcgc gcggcggagg ccgaccccgc 420

gagcaggaag ctcttcgtcc acggcctcgg ctggggcgcc ggagccgacg acctccgctc 480

cgcgttctcc cgcttcggcg agctcgagga ctgccgtgtc atctccgaca agcaatctgg 540

cagatctaag ggctacggct tcgtcctctt ccgctcccgc cgctccgcgc tccgcgccct 600

ccgccgcccc cagctccaaa tcggcggccg ccttgctttc tgccatctcg ctgcctcggg 660

tcctgcccct ccgacctccc agtcccagaa ccctagctcc aacaccaacg ccaactccgg 720

tgcggccaat aacgctggtt ctgcgtcgtc ttcacaacct gacaacatgc aacgcaaaat 780

ctttgttggt aacgtgcatg ctgatgtcga tgttgaccgc ctgtatgagt acttctcgca 840

atttggtgag attgaggagg ggccattggg atttgacaag accactggca agcctaaggg 900

gtttgcgctg ttcgtctaca agtcagtgga gagcgcccgc cgtgcgctgg aggagccaac 960

aaggaacttt gatggcaaaa tgctcaatgt gcagaaggct atagatggca ggaccaagaa 1020

cacacctggg atgaatgcaa attctaaccc tagtggtacc gctgcctcgg cagcagctgc 1080

tgctgcagct gcacagatga ctgctcctgc tactgctgca attacaccgt atgatgcatc 1140

agcttatggt gctactgccg ttcctgactt gggttatgcg cagcaagcgg ctatgttggg 1200

attgggtgca cagcagcagg cgtttgcaca acccaatgct gcaatgcttg ccatgatggc 1260

agcagctatg caaaacccag ctatgctcgc aacgctgaac cctgcttttg ctgctgctgc 1320

attgggtgct gggggccagc aggtgcacgc ggctggtatt ccaggttttg gagctcaggg 1380

ttttgggaca cagggctttg cagcaggtgc cgccgctttt ccaaatgcag cgggtgttca 1440

agctcctcct ggttttcagg gtccgcctgg gttccagact tctgctgggt ttcaggttgg 1500

ccaagcagct tcacaagcgg gtactgctgc ggctgctgcc gccggtgctg ctggttatca 1560

ggctgctgct gctgggcagg gccaagtgcc cggaacgcaa attgggggtg ctggttttca 1620

gggtggattt tgaaatcact aggtataaca attcacatcc tgcttaatcg cttcaagtcc 1680

tcatttacca ctaatgattt gagttagctt atcctccaaa tattattcgc aacttggtga 1740

tttgggctaa aaatttctga ttgtttctct atatcttagt ttggcatctg ctggtcgtga 1800

ttcgttcgtg tggttttgca ttcggatgga ttttgaatct atgttggtgc caaagtattg 1860

cgaaagggtt cctgctgttg gcttttacaa tcagatggct tttgaatcag gcaactttgt 1920

ggcagtattt acagaaggcg ctttcttgaa gaatttatgt tgtttttatt gcctactatg 1980

tagctttgtc cttgcaactt catcatgtac gttatggttt ctgtaatggg gtttaatgta 2040

ctgaaatatc tgtattttga cttgttggga tgttatccgg tcttattgat tatgtcaaat 2100

ggtttctggg cacttgaagg ctttttcttt tttgcagttg cagtgggatt tggaagaatg 2160

tgatattcat agcttttgcg ttcaccttgg aaatttctgg tttgaggctg agttcagctg 2220

ctatgacctt aagaccttga ttgccattga gctttatttc taaatctgca tagtgatctc 2280

ctgtagccaa ctagccatgg gaaataggtg aaagttcact ttgcgtttat aatctcttga 2340

aagagtataa attaatggat aatgcctcat cagcaaacac tgcatctcca ttggagattt 2400

tggaatgtgc ttttgttttt ctgataaagt aagtatctag aacatttcta taggattcaa 2460

gcatagtaat ggtactggcg ccctgtggac actgcatagg cttgactctt tctagtgcga 2520

tgtgctgatt gaaaagtgca tactagggtg taggtaatgt tcatcctgtc ataaactcat 2580

gcggactttt tagcttgtgc ttgctgtcag aatgtggtcc ctctaaatat gagctgtttt 2640

agcagaaggt tatggtctgc tgacagtgat ctttgctttg tgctgaggaa ggatagcatt 2700

caaaacattc atacagccca taaccatatg tgttctattt cttatatgca ccataagcgc 2760

aatcattaag tacggcagtt gaaacctttg ctttagagtt cttatcctat tgtctataga 2820

gtcgcaacaa ctatgcccat ccatcagatg tagatctgat tttcaaagtt gccccccttt 2880

ccaaatttgt cacaaaacca tctagtttca aggcttacaa cagtagatca agcacaccaa 2940

atggctttac attgctaaat gtcgtgaagt tattgtcact agctagaatt tgcgcatgat 3000

gaagttacag agactattga ggtctccgta agtttgctac atatcatttc cctgttattt 3060

tccatgtcat ggaagaaatc tccatctaat tgatcaactt tcacgttttg tctggtacaa 3120

ctaaattttt gttgtgagaa ctcgattagt caattatagg catcattctt gttacaaatg 3180

aacataatct ttcatatgtg gtggtaaaaa tctttacaat ataatcatta ctgctgaatt 3240

gttgtttata ttgttggatt tccttagtat catctttctt gaaatggatt tttttttgtt 3300

tcttctagtt gtgtatactg gcagcagaga cgaccagtta ttatttgatg atgattacat 3360

acttcatgta tccaatgggt atattgcttt atatctggca gtcactatga ttattacagt 3420

cttgtgtcta tttgccatca tgttctatct ccagttctct ttaccatatc actgcgtttg 3480

cattcttatc tttgaacgtg atgtcttgct aatttgagtt cttaagcgaa cttttcatat 3540

tttccatggg accatttcag tataaagact gcattgtgga atctggaaac tgttatttca 3600

taggggtccg tgattatgaa actttttctt ggatgcaatt tagcagttct ttaacctttt 3660

tccacacaat tctcacactt gtaatattag tggatttggg tatagtagct atgtgggagg 3720

tttggattct ttatcaattc gtttgcgctg caaaaatggc tagtgaactt aatcaccttt 3780

tggggccaga cgagtttcta caccgtctga ttttgtctct atatgtttgg tttttaagct 3840

gctgttctgc ttattgaagc accttttcac taagagactc caattggacg agggcacctt 3900

ttctagagcg ttcatgcgaa ttattttttg tggtgatcat ggggaatttg tttgtcactt 3960

accatgcaca gcatattaat gctctgaaag ctgctgtaac tcgggtgatg acaggcgaag 4020

gagctgtata tgccgtaaca tattaaac 4048

<210> 4

<211> 241

<212> DNA

<213> Artificial sequence

<400> 4

ctggttctgc gtcgtcttca caacctgaca acatgcaacg caaaatcttt gttggtaacg 60

tgcatgctga tgtcgatgtt gaccgcctgt atgagtactt ctcgcaattt ggtgagattg 120

aggaggggcc attgggattt gacaagacca ctggcaagcc taaggggttt gcgctgttcg 180

tctacaagtc agtggagagc gcccgccgtg cgctggagga gccaacaagg aactttgatg 240

g 241

<210> 5

<211> 1678

<212> DNA

<213> Artificial sequence

<400> 5

ctggttctgc gtcgtcttca caacctgaca acatgcaacg caaaatcttt gttggtaacg 60

tgcatgctga tgtcgatgtt gaccgcctgt atgagtactt ctcgcaattt ggtgagattg 120

aggaggggcc attgggattt gacaagacca ctggcaagcc taaggggttt gcgctgttcg 180

tctacaagtc agtggagagc gcccgccgtg cgctggagga gccaacaagg aactttgatg 240

ggagctctat cgcccctacg tcagctccat ctccaggtcc gtcgcttctc ttccatttct 300

tctcattttc gattttgatt cttatttctt tccagtagct cctgctctgt gaatttctcc 360

gctcacgata gatctgctta tactccttac attcaacctt agatctggtc tcgattctct 420

gtttctctgt ttttttcttt tggtcgagaa tctgatgttt gtttatgttc tgtcaccatt 480

aataataatg aactctctca ttcatacaat gattagtttc tctcgtctac aaaacgatat 540

gttgcatttt cacttttctt ctttttttct aagatgattt gctttgacca atttgtttag 600

atctttattt tattttattt tctggtgggt tggtggaaat tgaaaaaaaa aaaaacagca 660

taaattgtta tttgttaatg tattcatttt ttggctattt gttctgggta aaaatctgct 720

tctactattg aatctttcct ggatttttta ctcctattgg gtttttatag taaaaataca 780

taataaaagg aaaacaaaag ttttatagat tctcttaaac cccttacgat aaaagttgga 840

atcaaaataa ttcaggatca gatgctcttt gattgattca gatgcgatta cagttgcatg 900

gcaaattttc tagatccgtc gtcacatttt attttctgtt taaatatcta aatctgatat 960

atgatgtcga caaattctgg tggcttatac atcacttcaa ctgttttctt ttggctttgt 1020

ttgtcaactt ggttttcaat acgatttgtg atttcgatcg ctgaattttt aatacaagca 1080

aactgatgtt aaccacaagc aagagatgtg acctgcctta ttaacatcgt attacttact 1140

actagtcgta ttctcaacgc aatcgttttt gtatttctca cattatgccg cttctctact 1200

ctttattcct tttggtccac gcattttcta tttgtggcaa tccctttcac aacctgattt 1260

cccactttgg atcatttgtc tgaagactct cttgaatcgt taccacttgt ttcttgtgca 1320

tgctctgttt tttagaatta atgataaaac tattccatag tcttgagttt tcagcttgtt 1380

gattcttttg cttttggttt tctgcagaaa catgggtgca ggtggaagac gcgttaccca 1440

tcaaagttcc ttgttggctc ctccagcgca cggcgggcgc tctccactga cttgtagacg 1500

aacagcgcaa accccttagg cttgccagtg gtcttgtcaa atcccaatgg cccctcctca 1560

atctcaccaa attgcgagaa gtactcatac aggcggtcaa catcgacatc agcatgcacg 1620

ttaccaacaa agattttgcg ttgcatgttg tcaggttgtg aagacgacgc agaaccag 1678

Claims (10)

1. Use of DHT1 protein or a related biological material thereof for any of the following applications;

   p1, regulating and controlling plant types;

   p2, regulating and controlling the plant height;

   p3, regulating and controlling the plant ear length;

   p4, regulating and controlling the grain width of the plant;

   p5, regulating and controlling the tillering number of the plant;

   the DHT1 protein is any one of the following proteins:

   (A1) protein with an amino acid sequence of SEQ ID No. 1;

   (A2) the amino acid sequence shown in SEQ ID No.1 is substituted and/or deleted and/or added by one or more amino acid residues and is derived from the protein with the same function of rice;

   (A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and derived from rice having the same function;

   (A4) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of a protein defined in any one of (A1) to (A3);

   the related biological material is a nucleic acid molecule capable of expressing the DHT1 protein, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the nucleic acid molecule.
2. 2. Use according to claim 1, characterized in that: the expression level and/or activity of the DHT1 protein in the plant is reduced, and the plant height of the plant is reduced, the ear length of the plant is shortened, the grain width of the plant is shortened, and/or the tillering number of the plant is increased.
3. 3. Use of a substance capable of reducing the expression level and/or activity of DHT1 protein in a plant according to any one of (a1) - (a 4):

   (a1) reducing the plant height of the plant;

   (a2) reducing the ear length of the plant;

   (a3) reducing the grain width of the plant;

   (a4) increasing the number of tillers of the plant;

   the DHT1 protein is any one of the following proteins:

   (A1) protein with an amino acid sequence of SEQ ID No. 1;

   (A2) the amino acid sequence shown in SEQ ID No.1 is substituted and/or deleted and/or added by one or more amino acid residues and is derived from the protein with the same function of rice;

   (A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and derived from rice having the same function;

   (A4) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
4. 4. A method for producing a plant having a reduced plant height and/or a shortened panicle length and/or a reduced kernel width and/or an increased number of tillers, comprising the step of reducing the expression level and/or activity of DHT1 protein in a recipient plant;

   the DHT1 protein is any one of the following proteins:

   (A1) protein with an amino acid sequence of SEQ ID No. 1;

   (A2) the amino acid sequence shown in SEQ ID No.1 is substituted and/or deleted and/or added by one or more amino acid residues and is derived from the protein with the same function of rice;

   (A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and derived from rice having the same function;

   (A4) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
5. 5. A method for cultivating a transgenic plant with reduced plant height, shortened ear length, reduced kernel width and/or increased tiller number, comprising the steps of: inhibiting and expressing a nucleic acid molecule capable of expressing DHT1 protein in a receptor plant to obtain a transgenic plant; compared with the acceptor plant, the transgenic plant has reduced plant height, shortened spike length, reduced kernel width and/or increased tiller number;

   the DHT1 protein is any one of the following proteins:

   (A1) protein with an amino acid sequence of SEQ ID No. 1;

   (A2) the amino acid sequence shown in SEQ ID No.1 is substituted and/or deleted and/or added by one or more amino acid residues and is derived from the protein with the same function of rice;

   (A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and derived from rice having the same function;

   (A4) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
6. 6. The method of claim 5, wherein: the inhibition of the expression of the nucleic acid molecule capable of expressing the DHT1 protein in the recipient plant is achieved by introducing into the recipient plant an interference vector comprising a DNA fragment of formula I;

   SEQ_{forward direction}-X-SEQ_{Reverse direction}(I)；

   Said SEQ_{Forward direction}Is a partial fragment of SEQ ID No.2 or the full length thereof;

   said SEQ_{Reverse direction}And the sequence of SEQ_{Forward direction}Is complementary in reverse direction;

   said X is said SEQ_{Forward direction}And said SEQ_{Reverse direction}In the sequence, the X and the SEQ_{Forward direction}And said SEQ_{Reverse direction}Are not complementary.
7. 7. The method of claim 6, wherein: said SEQ_{Forward direction}Is the nucleotide sequence at position 650-890 of SEQ ID No. 2.
8. 8. Use or method according to any of claims 1-7, wherein: the nucleic acid molecule capable of expressing the DHT1 protein is any one of the following:

   (B1) a DNA molecule shown as SEQ ID No.2 or SEQ ID No. 3;

   (B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes said DHT1 protein;

   (B3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence defined in any one of (B1) - (B2) and encodes the DHT1 protein.
9. 9. Use of the method of any one of claims 4 to 8 in plant breeding.
10. 10. Use or method according to any of claims 1-9, wherein: the plant is a monocotyledon or a dicotyledon;

    further, the monocotyledon is a gramineous plant;

    further, the gramineous plant is a plant of the genus oryza;

    more specifically, the genus oryza plant is oryza sativa.

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